Correlation between gene expression and clinical scores in vernal keratoconjunctivitis
Authors: Andrea Leonardi, Fabiano Cavarzeran, Umberto Rosani, Giuseppe Righetti, Jean‐Sebastien Garrigue, Paola Brun
Journal: Allergy (2023)

Vernal keratoconjunctivitis (VKC) is a severe, chronic ocular allergic disease affecting mostly children and young adults, characterized by a Type 2 (T2) inflammation with possible corneal involvement and visual loss.1 Topical corticosteroids and calcineurin inhibitors, such as cyclosporine and tacrolimus, are required to treat moderate to severe cases not responding to conventional antiallergic drugs. However, long-term use of corticosteroids is contraindicated because of potential sight-threatening side effects. In patients resistant to topical immunomodulators, biologics may be an alternative option. Therefore, a better correlation between phenotypes and endotypes is needed. In this study we correlated the severity of the clinical manifestations with the local gene expression. Conjunctival epithelial samples from 15 VKC patients and 8 healthy subjects were collected, total RNA was extracted and further analyzed by Affymetrix platform (supplementary file).2 A severity score1-4 based on signs and symptoms was assigned to each patient3 while the ocular surface staining was assessed using the Oxford and the VKC-Collaborative Longitudinal Evaluation of Keratoconus (CLEK) scales (Table S1). The study complied with the tenets of the Declaration of Helsinki and was approved by the IRB of our Institution. Informed consent was obtained from all subjects (or parents), who were informed of the nature of the study. Dividing patients by the severity of the clinical score, 397 statistically significant differentially expressed genes (DEGs) were identified in patients with high score3, 4 but none in those with low score,1, 2 and 461 DEGs in patients with high VKC-CLEK score4-6 but none in those with low score (0–3). 297 DEGs were significantly overexpressed or downregulated in patients with high Oxford score3, 4 and 281 in those with low score (0–1). Therefore, gene expression changed with the severity of VKC. Looking at the top-100 DEGs in each patient, there were no clear differences from low1, 2 and high clinical scores3, 4 (Figure 1A,B). However, considering the extreme score values (1 vs. 4) there was a clear cut between overexpressed or downregulated genes (Figure 1C). Further analysis using Panther Classification System (http://pantherdb.org/) identified Gene Ontology Biological Processes (GOBP) that might be associated to higher and/or lower VKC severity (Figure 1D; Table S2). Inflammation mediated by chemokine and cytokine signaling, interleukin signaling, and nicotinic acetylcholine receptor signaling were the pathway hits that included three genes: IL-4Rα, IL-6, and TPR. Of the 41 downregulated genes, multiple GOBP and pathways were identified (Figure 1E). Considering the extreme scores of VKC-CLEK (0 vs. 6) and Oxford (0 vs. 4), of the overexpressed genes, 115 and 222 GOBP, and 11 and 34 pathways hits were identified, respectively (Figures S1 and S2). However, the relatively high number of genes overexpressed in each pathway did not allow to identify potential susceptible genes for corneal involvement in VKC. A cross-evaluation between the top 100 DEGs in each extreme severe scores showed that the clinical severity and VKC-CLEK shared 29 DEGs with interleukin signaling (IL-4Rα) as the only pathway hit. Desmoglein (DSG1), encoding for a protein related to epithelial barrier, and TIMP3, a metalloprotease inhibitor, were highly downregulated in both scales confirming that epithelial barrier dysfunction is associated with VKC4 and the relatively good correlation between the clinical and the VKC-CLEK scores. Beside gene expression, epigenetic repression and posttranscriptional and translational modifications can influence the fate of the described genes and pathways, making necessary dedicated studies to confirm our results. From these data, severe VKC patients may benefit by improving the epithelial barrier function and by targeting selectively T2 inflammation (IL-4R). Less clear is the role of TPR, which has a role in cell proliferation and in the autophagy machinery,5 which has been previously related to VKC.6 Even though topical cyclosporine and tacrolimus are commonly use for the treatment of severe VKC, biologics such as anti-IL-4R may further be considered for the management of severe cases. Andrea Leonardi (AL), Fabiano Cavarzeran (FC), Umberto Rosani (UR), Giuseppe Righetti (GR), Jean-Sébastien Garrigue (JSG), and Paola Brun (PB). Conception and design: AL, FC, and UR. Analysis and interpretation: AL, FC, UR, and PB. Writing the article: AL. Critical revision of the article: PB, UR, JSG, and GR. Final approval of the article: AL, UR, FC, GR, PD, JSG, and PB. Data Collection: AL, UR, FC, and GR. Provision of materials, patients, or resources: AL, JSG, and UR. Statistical expertise: FC. Obtaining funding: JSG, AL, and UR. Literature search: AL, PB, UR, and GR. Administrative, technical or logistic support: AL, FC, and JSG. None. Partially supported by an unrestricted grant from Santen SAS. Andrea Leonardi: No conflicts of interest. Fabiano Cavarzeran: No conflicts of interests. Umberto Rosani: No conflict of interest. Giuseppe Righetti: No conflicts of interests. Jean-Sébastien Garrigue: Santen SAS employee. Paola Brun: No conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1 Figure S2 Table S1 Table S2 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.